Method for screening antibody using patient-derived tumor spheroids

ABSTRACT

The present disclosure relates to a method of screening an antibody or an antigen-binding fragment thereof by use of patient-derived tumor spheroids, and more particularly to a method of screening an antibody or an antigen-binding fragment thereof, which binds specifically to an antigen, by use of patient-derived tumor spheroids containing the antigen.

TECHNICAL FIELD

The present disclosure relates to a method of screening an antibody oran antigen-binding fragment thereof by use of patient-derived tumorspheroids, and more particularly to a method of screening an antibody oran antigen-binding fragment thereof, which binds specifically to anantigen, by use of patient-derived tumor spheroids containing theantigen.

BACKGROUND ART

Antibody drugs are growing most rapidly in the field ofbiopharmaceuticals due to their high therapeutic effects and targetedtherapeutic properties. The antibody drug market which is thefastest-growing field among 200 biopharmaceuticals accounts for a highproportion (37%) of the total biopharmaceutical market, and the globalmarket for the antibody drugs is expected to grow at an average annualrate of 11.8% as US $51.5 billion in 2012, reaching US $89.9 billion in2017 (2013 Biopharmaceutics Trend Report, the Korean Ministry of Trade,Industry and Energy, 2013).

Diseases which are the major targets of antibody drugs are mostlyconcentrated in specific diseases such as such as intractable cancer(49%) and immune diseases (35%), and thus market competition amongproducts with similar indications is intense. Nevertheless, there aresubdivided therapeutic targets within diseases, so the field ofdevelopment of antibody therapeutic drugs for anticancer therapy isdetermined to continue to grow in the future.

The technology used to identify and secure antibody candidates which arethe major active ingredients of these antibody drugs may largely beclassified into the development of chimeric or humanized antibodiesusing hybridoma cell lines, methods employing transgenic mice, andmethods employing antibody display technology.

In 1975, a hybridoma technology was developed which produces monoclonalantibodies using hybrid cells formed by fusing cancer cells with normalcells. Since then, the active development of antibody drugs has started,and technologies have been developed which identify humanized antibodiesand human antibodies have been developed in order to solve the HAMA(human-anti-mouse antibody) reaction that occurs when mouse monoclonalantibodies are applied to humans.

The technical field for producing human antibodies can be exemplified bytransgenic mice and antibody display technologies (phage display, yeastdisplay, ribosome display, etc.). The development of antibodies usingtransgenic mice, which has recently been most frequently attempted, is atechnology that produces human monoclonal antibodies by applying theexisting hybridoma technology to transgenic mice transplanted with humanantibody genes. This technology has great advantages in that it canproduce a high-affinity antibody due to possible in vivo maturation andcan effectively produce a human antibody. However, it has disadvantagesin that the use conditions of transgenic mice are expensive and there isdifficulty in technical entry, such as production know-how.

Among antibody display technologies, phage display technologies aretechnologies of screening antibodies by displaying antibody fragments onthe surface of bacteriophages, and display technologies based on M13PIII phage are most widely used. However, these phase displaytechnologies have a difficulty in screening cell surface proteins (suchas G-protein receptor) or antibodies difficult to express recombinantly,because antibody fragments expressed by gene recombination are displayedon the surface of bacteriophages.

In order to solve these problems, a strategy that does not userecombinant proteins in the screening step has been developed, which isa strategy that uses cell surface proteins themselves for screening.

It has been found in conventional cited references that antibodies orpeptides that bind directly to antigens expressed on the cell surfacecan be screened by incubating antibody candidates with the cells(Andersen P S, et al., Proc Natl Acad Sci USA 93:1820-1824, 1996; BarryM A, et al., Nat Med 2:299-305, 1995; Cai X, Garen A. Proc Natl Acad SciUSA 92:6537-6541, 1995).

However, the above-described cell-based display method has adisadvantage in that because screening is performed using cells culturedin the laboratory, the antibody screened by the method cannot properlyexert its effect when actually applied to patients.

Meanwhile, an antibody-drug conjugate (ADC) is obtained by conjugating acytotoxic drug to an antibody via a linker. Since a monoclonal antibodyexhibits target specificity, the drug of the antibody-drug conjugate canbe delivered to a tumor expressing an antigen/target which is recognizedby a monoclonal antibody having selective targeting ability. Ideally,the antibody-drug conjugate which is maintained in a prodrug state inblood after administration should not be toxic, and as the antibody isinternalized into cancer cells after binding to its target tumorantigen, the drug is released in an active form and kills tumor cells.

Thus, determining a target/antigen to which an antibody binds is animportant starting point in the construction of antibody-drugconjugates. In particular, the target/antigen to which the antibodybinds has become a cell surface protein predominantly expressed(overexpressed) in tumor cells.

The definition of antigens that are expressed on the surface of humancancer cells means a broad range of targets that are overexpressedrelative to normal tissue or mutated and selectively expressed. The keyproblem is to identify appropriate antigens for antibody-basedtherapies. These therapeutic agents mediate changes in antigen orreceptor function (i.e., function as stimulants or antagonists),regulate the immune system through Fc and T cell activation, and exerttheir efficacy by delivering a specific drug bound to an antibody thattargets a specific antigen. Molecular technologies that can changeantibody pharmacokinetics, function, size and immunostimulationproperties have emerged as key elements in the development of newantibody-based therapies. Evidence obtained from clinical trials oftherapeutic antibodies on cancer patients emphasizes the importance ofapproaches for the affinity and binding ability between target antigensand antibodies, the selection of antibody structures, and the selectionof optimized antibodies, including therapeutic approaches (signalinginhibition or immune function).

Accordingly, the present inventors have made extensive efforts todevelop a method capable of screening antibodies highly effective forpatients, and as a result, have found that when an antibody library isscreened using patient-derived cells containing an antigen, an antibodycan be screened which has high sensitivity and accuracy and causes lessside effects, thereby completing the present disclosure.

DISCLOSURE OF INVENTION Technical Problem

An object of the present disclosure is to provide a method of screeningan antibody using patient-derived tumor spheroids, which overexpress anantigen.

Another object of the present disclosure is to provide a method ofscreening an antibody using a patient-derived tumor spheroidsoverexpressing an antigen and an animal model comprising the same.

Still another object of the present disclosure is to provide an antibodyor an antigen-binding fragment thereof, which is screened by the method.

Yet another object of the present disclosure is to provide a compositionfor preventing or treating cancer, which comprises, as an activeingredient, the antibody or antigen-binding fragment thereof, which isscreened by the method.

Technical Solution

To achieve the above object, the present disclosure provides a methodfor screening an antibody or an antigen-binding fragment thereof, whichbinds to an antigen, the method comprising the steps of: (i) treatingpatient-derived tumor spheroids, which express the antigen, with alibrary comprising antibodies or antigen-binding fragments thereof, andscreening antibodies or antigen-binding fragments thereof which bind tothe antigen; (ii) incubating the screened antibodies or antigen-bindingfragments thereof with patient-derived tumor spheroids that do notexpress the antigen; and (iii) separating or removing antibodies orantigen-binding fragments thereof, which bind to the patient-derivedtumor spheroids of step (ii), from the antibodies or antigen-bindingfragments thereof screened in step (i).

The present disclosure also provides a method for screening an antibodyor an antigen-binding fragment thereof, which binds to an antigen, themethod comprising the steps of: (i) treating patient-derived tumorspheroids, which express the antigen, with a library comprisingantibodies or antigen-binding fragments thereof, and performing firstscreening of antibodies or antigen-binding fragments thereof which bindto the antigen; (ii) treating patient-derived tumor spheroids, which donot express the antigen, with the first screened antibodies orantigen-binding fragments thereof; (iii) administering antibodies orantigen-binding fragments thereof, obtained by separating or removingantibodies or antigen-binding fragments thereof that binds to thepatient-derived tumor spheroids of step (ii) from the antibodies orantigen-binding fragments thereof screened in step (i), to animal modelstransplanted with the patient-derived tumor spheroids which express theantigen, and performing second screening of antibodies orantigen-binding fragments which bind to the antigen; and (iv) separatingor removing antibodies or antigen-binding fragments thereof, which bindto an antigen other than the antigen, from the second screenedantibodies or antigen-binding fragments thereof.

The present disclosure also provides an antibody or an antigen-bindingfragment thereof, which is screened by the screening method.

The present disclosure also provides a composition for preventing ortreating cancer, which comprises the antibody or the antigen-bindingfragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a conceptual view showing a method of the present disclosure.

FIG. 2 schematically shows a method of screening an antibody that bindsto neuropilin 1 (NRP1) antigen, according to one embodiment of thepresent disclosure.

FIG. 3 schematically shows a method for in vivo screening of an antibodythat binds to NRP1 antigen.

FIG. 4 shows the configuration of a phagemid vector for production of ananti-NRP1 antibody fragment according to one embodiment of the presentdisclosure.

FIG. 5 shows the results of Coomassie staining of each anti-NRP1antibody fragment purified according to one example of the presentdisclosure.

FIG. 6 shows the ELISA results indicating the binding affinities ofthree anti-NRP1 antibody fragments, screened according to one example ofthe present disclosure, for NRP1.

FIG. 7 shows the ELISA results indicating the binding affinities ofthree anti-NRP1 antibody fragments, screened according to one example ofthe present disclosure, according to NRP1 concentration.

FIG. 8 shows the results of SPR analysis performed to analyze the KDvalues of three anti-NRP1 antibody fragments, screened according to oneexample of the present disclosure.

FIG. 9 shows the FACS analysis results indicating the binding affinitiesof anti-NRP1 antibody fragments, screened according to one example ofthe present disclosure, for patient-derived tumor spheroidsoverexpressing NRP1.

FIGS. 10a to 10c are confocal laser scanning micrographs showing theinternalizing function of three anti-NRP1 antibody fragments screenedaccording to one example of the present disclosure.

FIG. 11 shows the results of confirming the binding epitope of anti-NRP1antibody fragments screened according to one example of the presentdisclosure.

FIG. 12 shows the results of RNA-seq analysis performed to screen a cellline overexpressing NRP1.

FIG. 13 shows purity for three anti-NRP1 IgG antibodies.

FIG. 14 shows the results of an endotoxin test for three anti-NRP1 IgGantibodies.

FIG. 15 shows the results of measuring the KD values of anti-NRP1 IgGantibodies by ELISA.

FIG. 16 shows the specific binding affinities of three anti-NRP1 IgGantibodies for human NRP1.

FIG. 17 shows the results of confirming that three anti-NRP1 IgGantibodies screened according to a method of the present disclosure areinternalized into patient-derived cancer cells.

FIG. 18 shows the results of confirming that three anti-NRP1 IgGantibodies screened according to a method of the present disclosure showcancer cell-specific internalization.

FIG. 19 shows the results of confirming that three anti-NRP1 IgGantibodies screened according to a method of the present disclosure showa higher difference between their binding affinities for normal cellsand cancer cells than known NRP1 antibody.

FIG. 20 shows the results of confirming that three anti-NRP1 IgGantibodies screened according to a method of the present disclosureexhibit an excellent effect of inhibiting cancer cell migration.

FIG. 21 shows the results of confirming that an anti-NRP1 IgG antibodyscreened according to a method of the present disclosure exhibits anexcellent effect of inhibiting cancer cell migration.

FIG. 22 shows the results of confirming the change in signalingsubstances by an anti-NRP1 IgG antibody screened according to a methodof the present disclosure.

FIG. 23 shows the results of a TUNEL assay performed to confirm thatapoptosis is increased by an anti-NRP1 IgG antibody screened accordingto a method of the present disclosure.

FIG. 24 shows the results of evaluating the efficacy of an anti-NRP1 IgGantibody, screened according to a method of the present disclosure,against glioblastoma.

FIG. 25 shows the results of evaluating the efficacy of an anti-NRP1 IgGantibody, screened according to a method of the present disclosure,against lung cancer.

FIG. 26 shows the results of confirming the glioblastoma-specificbinding of an anti-NRP1 IgG antibody, screened according to a method ofthe present disclosure.

FIG. 27 shows the results of evaluating the distribution of an anti-NRP1IgG antibody, screened according to a method of the present disclosure,in normal tissues.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all the technical and scientific terms usedherein have the same meaning as those generally understood by one ofordinary skill in the art to which the invention pertains. Generally,the nomenclature used herein and the experiment methods, which will bedescribed below, are those well-known and commonly employed in the art.

In order to screen an antibody that has a high possibility of success infuture clinical trials and can effectively act in cells afterinternalization, the present inventors have made extensive efforts todevelop an anticancer therapeutic antibody using patient-derived tumorspheroids containing an antigen. As a result, the present inventors havescreened an antibody that binds to NRP1 antigen with high affinity andis internalized into cells using phage display technology, and haveconfirmed that such an antibody is internalized into cells.

Accordingly, in one example of the present disclosure, in order toscreen an antibody which is internalized into cells by binding to NRP1(neuropilin-1) known to be expressed in various cancers, phage displaybased on glioblastoma patient-derived tumor spheroids was performed. Asa result, it was confirmed that the screened anti-NRP1 antibody wasinternalized into cells after binding to NRP1 expressed on the cancercell surface (FIGS. 10a to 10c ), and that the binding epitope of theantibody did differ from that of a conventional antibody (FIG. 11).

Therefore, in one aspect, the present disclosure is directed to a methodfor screening an antibody or an antigen-binding fragment thereof, whichbinds to an antigen, the method comprising the steps of: (i) treatingpatient-derived tumor spheroids, which express the antigen, with alibrary comprising antibodies or antigen-binding fragments thereof, andscreening antibodies or antigen-binding fragments thereof which bind tothe antigen; (ii) incubating the screened antibodies or antigen-bindingfragments thereof with patient-derived tumor spheroids that do notexpress the antigen; and (iii) separating or removing antibodies orantigen-binding fragments thereof, which bind to the patient-derivedtumor spheroids of step (ii), from the antibodies or antigen-bindingfragments thereof screened in step (i).

As used herein, the term “expression” means a process in which anantigen is produced from a structural gene, and includes a process inwhich a gene is transcribed into mRNA which is then translated into anantigen. Generally, a specific antigen can contribute to the creation ofdisease, for example, cancer, and may be overexpressed to inhibit theapoptosis of, for example, cancer cells, or overexpression of theantigen can increase the invasiveness or migration of, for example,cancer cells. Thus, “expression of the antigen” in the method forscreening the antibody or antigen-binding fragment thereof which bindsto the antigen, according to the present disclosure, may be meant toinclude the overexpression or abnormal activation of the antigen.

As used herein, the term “antibody” is an immunoglobulin selected fromthe group consisting of IgA, IgE, IgM, IgD, IgY and IgG, which may bindspecifically to a target antigen. The antibody is composed of two lightchains and two heavy chains, and each of the chains is composed of avariable domain, which has a variable amino acid sequence, and aconstant domain which has a constant amino acid sequence. At the end ofthe three-dimensional structure of the variable region, anantigen-binding domain is located. This antigen-binding domain iscomposed of complementarity determining regions, and each of the lightand heavy chains comprises three complementarity determining regions.The complementarity determining regions have an especially high aminoacid sequence variability among the variable domains. Due to this highvariability, specific antibodies for various antigens can be found. Thescope of the present disclosure also includes an intact antibody form aswell as an antigen-binding fragment of the antibody molecule.

As used herein, the term “ScFv (single-chain Fv)” is an antibodyconsisting of light chain and heavy chain variable domains linkedtogether. In some cases, the ScFv may comprise a linker consisting ofpeptide chains having about 15 amino acids linked together. In thiscase, the ScFv may have a structure of light chain variabledomain-linker-heavy chain variable domain, or a structure of heavy chainvariable domain-linker-light chain variable domain, and has antigenspecificity equal or similar to that of the parent antibody.

The complete antibody is a structure having two full-length light chainsand two full-length heavy chains, and each light chain is linked by adisulfide bond with a heavy chain. A constant region of the heavy chainhas gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types.Sub-classes have gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4),alpha 1 (α1), and alpha 2 (α2) types. A constant region of the lightchain has kappa (κ) and lambda (λ) types.

An antigen-binding fragment or an antibody fragment of an antibodyrefers to a fragment having an antigen-binding function and includesFab, F(ab′), F(ab′)2, Fv, and the like. Fab of the antibody fragmentshas a structure including variable regions of a light chain and a heavychain, a constant region of the light chain, and a first constant region(CH1) of the heavy chain with one antigen-binding site. Fab′ differsfrom Fab in that it has a hinge region containing one or more cysteineresidues at the C-terminal of the heavy chain CH1 domain. The F(ab′)2antibody is produced when the cysteine residue of the hinge region ofthe Fab′ forms a disulfide bond. Recombinant techniques for generatingFv fragments with minimal antibody fragments having only a heavy chainvariable region and a light chain variable region are described in PCTInternational Publication Nos. WO88/01649, WO88/06630, WO88/07085,WO88/07086, and WO88/09344. A two-chain Fv has a non-covalent bondingbetween a heavy chain variable region and a light chain variable region.A single chain Fv (scFv) is connected to a heavy chain variable regionand a light chain variable region via a peptide linker by a covalentbond or directly at the C-terminal. Thus, the single chain Fv (scFv) hasa structure such as a dimer like the two-chain Fv. Such an antibodyfragment can be obtained using a protein hydrolyzing enzyme (forexample, when the whole antibody is cleaved with papain, Fab can beobtained, and when whole antibody is cut with pepsin, F(ab′)2 fragmentcan be obtained), and it can also be produced through gene recombinanttechnology.

As used herein, the term “antibody (or ScFv) library” is a collection ofvarious antibody genes having different sequences. To separate anantibody specific for any antigen from an antibody library, a very highdiversity is required. A library consisting of different antibody clonesis constructed and used. The antibody gene of this antibody library maybe cloned into, for example, a phagemid vector, and transformed into ahost cell (E. coli).

As used herein, the term “nucleic acid” may be used interchangeably withthe term “gene” or “nucleotide”. For example, the nucleic acid may beselected from the group consisting of natural/synthetic DNA, genomicDNA, natural/synthetic RNA, cDNA and cRNA, but is not limited thereto.

As used herein, the term “phagemid” vector is a plasmid DNA which isused in phage display and has a phage origin of replication. Itgenerally has an antibiotic resistance gene as a selection marker. Aphagemid vector that is used in phage display includes the gIII gene ofM13 phage or a portion thereof, and the ScFv gene is ligated to the 5′end of the gIII gene and expressed in a host cell.

As used herein, the term “helper phage” is a phage that providesnecessary genetic information so that phagemid is packaged into phageparticles. Since phagemid includes the gIII gene of phage or a portionthereof, a host cell (transformant) transformed with the phagemid isinfected with the helper phage to provide the remaining phage gene. Thehelper phages include M13K07 or VCSM13, and mostly include an antibioticresistance gene such as a kanamycin resistance gene so that atransformant infected with the helper phage can be selected. Inaddition, the packaging signal is defective, and thus the phagemid generather than the helper phage gene is selectively packaged into phageparticles.

As used herein, the term “signal sequence” is either a nucleotidesequence which is located at the 5′end of the gene and functions as anecessary signal when a protein encoded by the gene is secretedextracellularly, or an amino acid sequence corresponding thereto.

As used herein, the term “Phage display” refers to a technique thatdisplays a fusion protein by fusing a mutant polypeptide and at least apart of a coat protein on a surface of phase, for example, a fibrousphage particle. The phage display is useful for targeting a largelibrary of randomized protein variants to quickly and efficientlyclassify sequences that bind to target antigens with high affinity.Displaying peptides and protein libraries on phage has been used toscreen millions of polypeptides to identify polypeptides with specificbinding properties.

The phage display technique has provided a powerful tool for generatingand screening novel proteins that bind to specific ligands (e.g.,antigens). Using the phage display technique, a large library of proteinvariants can be generated and sequences binding to the target antigenswith high affinity can be rapidly classified. The nucleic acid encodingthe mutant polypeptide is fused with a nucleic acid sequence encoding aviral coat protein, e.g., a gene III protein or a gene VIII protein. Amonovalent phage display system has been developed in which a nucleicacid sequence encoding a protein or polypeptide is fused with a nucleicacid sequence encoding a part of the gene III protein. In the monovalentphage display system, the gene fusion is expressed at a low level, andthe wild-type gene III protein is also expressed, thereby maintainingthe infectivity of the particles.

Demonstrating the expression of peptides on the fibrous phage surfaceand the expression of functional antibody fragments in the peripheralcytoplasm of a host cell is important in developing antibody phagedisplay libraries. Libraries of antibodies or antigen-bindingpolypeptides have been prepared in a number of ways, for example byaltering a single gene by inserting a random DNA sequence or by cloninga related genic line. The library can be screened for expression ofantibodies or antigen binding proteins with the desired characteristics.

The phage display technique has several advantages over conventionalhybridomas and recombinant methods for producing antibodies with thedesired characteristics. This technique allows the generation of a largeantibody library having various sequences in a short time without theuse of animals. The production of hybridomas or humanized antibodies maytake several months to manufacture. Further, the phage antibody librarymay produce antibodies against antigens that are toxic or have lowantigenicity since no immunity is required. The phage antibody librarycan also be used to generate and identify novel therapeutic antibodies.

A technology can be used in which human antibodies are generated fromvirgin B-cell Ig repertoires or human germline sequences immunized ornon-immunized using a phage display library. Various lymphatic tissuesmay be used to prepare virgin or non-immune antigen-binding libraries.

Techniques for identifying and separating high affinity antibodies froma phage display library are important for separating new therapeuticantibodies. The separation of high affinity antibodies from the librarymay depend on the size of the library, production efficiency inbacterial cells, and library diversity. The size of the library isreduced by inefficient production due to improper folding of an antibodyor antigen binding protein and the presence of the stop codon.Expression in bacterial cells can be inhibited when an antibody orantigen binding domain is not properly folded. The expression can beincreased by alternately mutating residues on a surface of avariable/constant interface or selected CDR residues. A sequence of theframework region is one element to provide appropriate folding whenantibody phage libraries are generated in bacterial cells.

It is important to generate various libraries of an antibody or antigenbinding proteins in high affinity antibody separation. The CDR3 regionhas been found to often participate in antigen binding. The CDR3 regionon a heavy chain varies considerably in terms of size, sequence, andstructural steric conformation so that various libraries can be preparedusing the CDR3 region.

Further, diversity may be generated by randomizing the CDR regions ofthe variable heavy and light chains using all amino acids at eachposition. The use of all 20 amino acids results in an increasedvariability of variant antibody sequences and an increased chance ofidentifying new antibodies.

As used herein, the term “neuropilin” or “NRP” collectively includesneuropilin-1 (NRP1), neuropilin-2 (NRP2), and their isoforms andvariants. Neuropilins are 120-130 kDa non-tyrosine kinase receptors.There are multiple NRP-1 and NRP-2 splice variants and soluble isoforms.The basic structure of neuropilins comprises five domains: threeextracellular domains (ala2, blb2 and c), a transmembrane domain, and acytoplasmic domain. The ala2 domain is homologous to complementcomponents Clr and Cls (CUB), which generally contains four cysteineresidues that form two disulfide bridges. The blb2 domain is homologousto coagulation factors V and VIII. The central portion of the c domainis designated as MAM due to its homology to meprin, AS and receptortyrosine phosphotase μ proteins. The ala2 and blb2 domains areresponsible for ligand binding, whereas the c domain is critical forhomodimerization or heterodimerization.

“Neuropilin-mediated biological activity” refers to physiological orpathological events in which neuropilin-1 plays a substantial role. Forexample, such activities may be axon guidance during embryonic nervoussystem development or neuron-regeneration, angiogenesis (includingvascular modeling), tumorgenesis and tumor metastasis, but are notlimited thereto.

The antibody of the present disclosure includes monoclonal antibodies,multispecific antibodies, human antibodies, humanized antibodies,chimeric antibodies, and the like, but is not limited thereto. Theantibody of the present disclosure includes an antigen-binding fragmentof the antibody or an antibody fragment, and the fragment may includesingle-chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFV), and anti-idiotype (anti-Id)antibodies.

The monoclonal antibody refers to an antibody obtained from asubstantially homogeneous population of antibodies, i.e., the sameexcept for possible naturally occurring mutations that may be present intrace amounts of individual antibodies that occupy the population. Themonoclonal antibody is highly specific and is derived against a singleantigenic site.

The non-human (e.g. murine) antibody of the “humanized” form is achimeric antibody containing minimal sequence derived from non-humanimmunoglobulin. In most cases, the humanized antibody is a humanimmunoglobulin (receptor antibody) that has been replaced by a residuefrom the hypervariable region of a non-human species (donor antibody),such as a mouse, rat, rabbit, and non-human primate, having specificity,affinity, and ability to retain a residue from the hypervariable regionof the receptor.

“Human antibody” is a molecule derived from human immunoglobulin andmeans that all of the amino acid sequences constituting the antibodyincluding the complementarity determining region and the structuralregion are composed of human immunoglobulin.

A heavy chain and/or light chain is partly identical or homologous tothe corresponding sequence in an antibody derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remaining chain(s) are identical or homologous to correspondingsequences in an antibody derived from another species or belonging toanother antibody class or subclass “chimeric” antibodies(immunoglobulins) as well as a fragment of such antibody exhibiting thedesired biological activity.

“Antibody variable domain” as used herein refers to the light and heavychain regions of an antibody molecule including the amino acid sequencesof a complementarity determining region (CDR; i.e., CDR1, CDR2, andCDR3) and a framework region (FR). VH refers to a variable domain of theheavy chain. VL refers to a variable domain of the light chain.

“Complementarity determining region” (CDR; i.e., CDR1, CDR2, and CDR3)refers to the amino acid residue of the antibody variable domain, whichis necessary for antigen binding. Each variable domain typically hasthree CDR regions identified as CDR1, CDR2, and CDR3.

“Framework region” (FR) is a variable domain residue other than a CDRresidue. Each variable domain typically has four FRs identified as FR1,FR2, FR3, and FR4.

The method of the present disclosure comprises step (i) of treatingpatient-derived tumor spheroids, which express the antigen, with alibrary comprising antibodies or antigen-binding fragments thereof, andscreening antibodies or antigen-binding fragments thereof which bind tothe antigen.

The patient-derived tumor spheroids may be, for example, cancerpatient-derived tumor spheroids, and cancer patient-derived cancer cellsexhibit physiological characteristics different from those of normalcells. Such physiological characteristics are determined by a geneexpression pattern in which the expression of the antigen increases ordecreases specifically in cancer cells compared to normal cells. Thisgene expression pattern may be patient-specific, or may show a patient'stissue-specific difference.

In the present disclosure, the antigen may be an antigen which isinvolved in the development, growth and migration of cancer or a tumor.The antigen may be in the form of an oligomer, a peptide, a polypeptideor a protein.

A method for measuring the antigen contents of the patient-derived tumorspheroids and normal cells may comprise measuring and comparing theexpression level of a gene or protein encoding the antigen. Preferably,the method may be performed by any one method selected from the groupconsisting of FACS, ELISA, whole exome sequencing, and RNA sequencing,but is not limited thereto.

In the present disclosure, the patient-derived tumor spheroid may bederived from a solid cancer patient, and the solid cancer may beselected from the group consisting of liver cancer, glioblastoma,ovarian cancer, colon cancer, head and neck cancer, bladder cancer,renal cell cancer, breast cancer, metastatic cancer, prostate cancer,pancreatic cancer, and lung cancer, but is not limited thereto.

In the present disclosure, the library is a collection of antibodiesand/or antigen-binding fragments thereof, may be displayed forscreening, and may be constructed as full-length antibodies. Theantibodies and/or antigen-binding fragments thereof in the library maybe displayed on, for example, ribosomes, phages, or cells.

A method for obtaining the patient-derived tumor spheroids is notparticularly limited, and may, for example, comprise the steps of: (a)dissociating isolated cancer patient-derived cancer tissue, andcollecting a cell fraction from the dissociated tissue; and (b) treatingthe collected cell fraction with protease, followed by filtration,centrifugation and suspension, thereby obtaining single cells.

In the present disclosure, the protease may be an enzyme capable ofperforming proteolysis, and examples thereof may include: endopeptidasethat degrades a protein by protein catabolism that hydrolyzes a peptidebond connecting amino acids in the protein; and exopeptidase thathydrolyzes a peptide bond from the N-terminus or C-terminus of aprotein.

The method of the present disclosure also comprises step (ii) ofincubating the screened antibodies or antigen-binding fragments thereofwith patient-derived tumor spheroids that do not express the antigen.

Step (ii) is a step of performing negative selection on the antibodiesor antigen-binding fragments thereof screened in step (i). The method ofthe present disclosure also comprises step (iii) of separating orremoving antibodies or antigen-binding fragments thereof, which bind tothe patient-derived tumor spheroids of (ii), from the antibodies orantigen-binding fragments thereof screened in (i). By virtue of thisstep, an antibody having high selectivity for an antigen can bescreened.

In the present disclosure, the patient-derived tumor spheroids whichdoes not express the antigen, which are used in step (ii), may be cellsthat do not naturally express the antigen, or patient-derived tumorspheroids artificially engineered so as not to express the antigen. Amethod for artificially engineering the cells may be any method thatprevents the antigen from being expressed. Preferably, thepatient-derived tumor spheroids that do not express the antigen may beobtained by treatment with one or more selected from the groupconsisting of aptamers, siRNA, single-stranded siRNA, microRNA, andshRNA, which bind to the antigen.

In the present disclosure, the step of screening only an antibody, whichbinds to the antigen, by use of the patient-derived tumor spheroids thatdo not express the antigen, is a negative selection step which isperformed following the immediately preceding positive selection step,and thus has the effect of increasing the accuracy of the screenedantibody for the antigen.

In some cases, the method of the present disclosure may furthercomprise, during or after step (ii), a step of performing phage displayat 4° C., and then screening an antibody, which is internalized into thecells, at an increased temperature of 37° C. By virtue of this step, theantibody internalized into the cells can be screened.

The separating or removing of the antibodies or antigen-bindingfragments thereof may be performed by using electrophoresis,centrifugation, gel filtration, precipitation, dialysis, chromatography(including ion exchange chromatography, affinity chromatography,immunoadsorption chromatography, size exclusion chromatography, and thelike), isoelectric focusing, and variations and combinations thereof,but is not limited thereto.

The separating or removing of the antibodies or antigen-bindingfragments thereof may be performed by removing impurities by, forexample, centrifugation or ultrafiltration, and purifying the resultingproduct by using, for example, affinity chromatography and the like.Other additional purification techniques, for example, anion or cationexchange chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, and the like may be used.

In another aspect, the present disclosure is directed to a method forscreening an antibody or an antigen-binding fragment thereof, whichbinds to an antigen, the method comprising the steps of: (i) treatingpatient-derived tumor spheroids, which express the antigen, with anantibody library, and performing first screening of antibodies orantigen-binding fragments thereof which bind to the antigen; (ii)treating patient-derived tumor spheroids, which do not express theantigen, with the first screened antibodies or antigen-binding fragmentsthereof; (iii) administering antibodies or antigen-binding fragmentsthereof, obtained by separating or removing antibodies orantigen-binding fragments thereof that binds to the patient-derivedtumor spheroids of step (ii) from the antibodies or antigen-bindingfragments thereof screened in step (i), to animal models transplantedwith the patient-derived tumor spheroids which express the antigen, andperforming second screening of antibodies or antigen-binding fragmentswhich bind to the antigen; and (iv) separating or removing antibodies orantigen-binding fragments thereof, which bind to an antigen other thanthe antigen, from the second screened antibodies or antigen-bindingfragments thereof.

The description of the same configuration as that described in theabove-described screening method can be applied equally.

In another example of the present disclosure, in order to screen anantibody which is internalized into cells by binding to NRP1(neuropilin-1) known to be expressed in various cancers, phage displaybased on glioblastoma patient-derived tumor spheroids was performed, andthen the first screened antibody candidates were injected intoimmunodeficient mice including the patient-derived tumor spheroids, andsecond screening in vivo was performed. As a result, it was confirmedthat the screened anti-NRP1 antibody was internalized into cells afterbinding to NRP1 expressed on the cancer cell surface (FIGS. 9a to 9c ),and that the binding epitope of the antibody did differ from that of aconventional antibody (FIG. 10).

In particular, the method of the present disclosure further comprises astep of administering antibodies or antigen-binding fragments thereof,obtained by separating or removing antibodies or antigen-bindingfragments thereof that binds to the patient-derived tumor spheroids ofstep (ii) from the antibodies or antigen-binding fragments thereofscreened in step (i), to animal models transplanted with thepatient-derived tumor spheroids which overexpress the antigen, andperforming second screening of antibodies or antigen-binding fragmentswhich bind to the antigen.

The screening method comprising the above-described steps according tothe present disclosure makes it possible to sufficiently reflect thecharacteristics of patients through animal models transplanted withpatient-derived tumor spheroids overexpressing the antigen. Thus, it maybe used for the screening of a patient-specific therapeutic drug and theselection of a treatment method, and can also screen an antibody or anantigen-binding fragment thereof, which is particularly suitable forpatient characteristics, with high accuracy.

In the present disclosure, the animal models transplanted withpatient-derived tumor spheroids overexpressing the antigen may be anyanimals including the patient-derived tumor spheroids. Preferably, theanimal models may be immunodeficient mice. The “immunodeficient mice”refer to mice generated by artificially damaging some elements of theimmune system at the gene level such that the immune system becomesabnormal and glioblastoma can develop. Most preferably, theimmunodeficient mice may be nude mice, NOD (non-obese diabetic) mice,SCID (Severe combined immunodeficiency) mice, NOD-SCID mice, or NOG(NOD/SCID I12rg−/−) mice.

The method according to the present disclosure can screen, for example,an antibody that binds to NRP1 overexpressed in patient-derived tumorspheroids, but is not limited thereto. The antibody may be, for example,an antibody that binds to the VEGF165 domain of NRP1, but the bindingepitope of this antibody may differ from that of a conventional antibody(MNRP1685A, antibody, Genetech) known to bind to the VEGF165 domain ofNRP1.

The antibody or antigen-binding fragment thereof screened by the methodaccording to the present disclosure may be, for example, an IgG format,an Fab′ fragment, an F(ab′)2 fragment, an Fab fragment, an Fv fragment,or a single-chain Fv (scFv) fragment. Preferably, it may be converted toan IgG format.

In still another aspect, the present disclosure is directed to anantibody or an antigen-binding fragment thereof, which is screened bythe method.

An antibody or antibody fragment of the present disclosure may include,within the scope of specifically recognizing NRP1, the sequence of theanti-NRP1 antibody of the present disclosure described herein as well asbiological equivalents thereof. The amino acid sequence of the antibodymay be additionally modified to further improve the binding affinityand/or other biological properties of the antibody. Such modificationsinclude, for example, deletion, insertion and/or substitution of theamino acid sequence residues of the antibody. Such amino acid variationsare made based on the relative similarity of amino acid side chainsubstituents, such as hydrophobicity, hydrophilicity, charge, and size.By analysis of the size, shape and type of amino acid side chainsubstituents, it is recognized that each of arginine, lysine andhistidine is a positively charged residue; alanine, glycine and serinehave similar sizes; and phenylalanine, tryptophan and tyrosine havesimilar shapes. Based on these considerations, it is thus found thatarginine, lysine and histidine; alanine, glycine and serine; andphenylalanine, tryptophan and tyrosine, respectively, are biologicallyfunctional equivalents.

In introduction of mutations, the hydropathic index of amino acids canbe considered. Each amino acid is assigned a hydrophobic index accordingto its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The hydrophobic amino acid index is very important in imparting theinteractive biological function of proteins. It is well known thatsubstitution with an amino acid having a similar hydrophobic index canretain similar biological activities. When a mutation is introduced withreference to a hydrophobic index, the substitution is made between aminoacids showing a hydrophobic index difference preferably within ±2, morepreferably within ±1, even more preferably within ±0.5.

Meanwhile, it is also well known that the substitution between aminoacids with similar hydrophilicity values leads to proteins withequivalent biological activity. As disclosed in U.S. Pat. No. 4,554,101,the following hydrophilicity values are assigned to each amino acidresidue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); andtryptophan (−3.4).

Amino acid substitution in proteins that do not totally alter theactivity of the molecule is known in the art. The substitution occursthe most commonly between amino acid residues, e.g., Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Considering the mutation having the above-mentioned biologicalequivalent activity, the antibody of the present disclosure or thenucleic acid molecule encoding the same is interpreted to include asequence showing substantial identity with the sequence described in thesequence listing. The substantial identity means a sequence showing atleast 61% homology, more preferably 70% homology, even more preferably80% homology, and most preferably 90% homology by aligning the sequenceof the present disclosure with any other sequence as much as possibleand analyzing the aligned sequence using algorithms commonly used in theart. Alignment methods for sequence comparison are well known in theart. NCBI Basic Local Alignment Search Tool (BLAST) may be accessiblefrom, e.g., NBCI and can be used in association with sequence analysisprograms such as blastp, blasm, blastx, tblastn and tblastx on theInternet. BLSAT is available athttps://blast.ncbi.nlm.nih.gov/Blast.cgi. A comparison of sequencehomology using this program can be found athttps://blast.ncbi.nlm.nih.gov/Blast.cgi.

In the present disclosure, the antibody or antigen-binding fragmentthereof may bind to an antigen, for example, an antigen involved indevelopment, growth and migration of cancer or tumor. Form of theantigen may be, for example, in the form of oligomer, peptide,polypeptide or protein.

In one example of the present disclosure, the present disclosure mayprovide a method for screening an antibody or an antigen-bindingfragment thereof, which binds specifically to NRP1.

In yet another aspect, the present disclosure is directed to acomposition for preventing or treating cancer, which comprises theantibody or antigen-binding fragment thereof as an active ingredient.

The present disclosure may be, for example, a pharmaceutical compositionfor preventing or treating cancer, comprising (a) a pharmaceuticaleffective amount of an antibody or antigen-binding fragment thereofagainst NRP1 according to the present disclosure; and (b) apharmaceutically acceptable carrier. The present disclosure is alsodirected to a method for prevention or treatment of cancer, comprisingadministering an effective amount of an antibody or antigen-bindingfragment thereof against NRP1 according to the present disclosure to apatient.

Since the composition uses the anti-NRP1 antibody or antigen-bindingfragment thereof of the present disclosure as an active ingredient, thedescriptions common to both of them are excluded in order to avoid theexcessive complexity of the present specification caused by the repeateddescriptions.

As demonstrated in Examples as described below, the anti-NRP1 antibodyof the present disclosure can inhibit the migration of cancer cellsoverexpressing NRP1. As such, the antibody and antigen-binding fragmentthereof of the present disclosure binds to NRP1 with high affinity andthus inhibits the migration of cancer cells overexpressing NRP1, so thatit can be used in the prevention and treatment of a cancer.

In one example of the present disclosure, it was confirmed that theanti-NRP1 antibody screened by the method of the present disclosurecould show cancer cell-specific internalization (Example 9), and couldexhibit the effect of increasing apoptosis and a desired tumor growthinhibitory effect in solid cancers, for example, glioblastoma and lungcancer (Example 11). In particular, it was confirmed that the anti-NRP1antibody screened by the method of the present disclosure had little orno binding affinity for normal tissue, suggesting that the side effectsof the antibody can be minimized (Example 12).

As used herein, the term “prevention” means any action that inhibits ordelays progress of a cancer by administration of a composition accordingto the present disclosure, and “treatment” means suppression ofdevelopment, alleviation, or elimination of a cancer.

The composition is applied to a disease that is a cancer overexpressingNRP1, for examples, glioblastoma, astrocytoma, glioma, neuroblastoma,testicular cancer, colon cancer, melanoma, pancreatic cancer, lungcancer, breast cancer, esophageal cancer, and prostate cancer.

As used herein, the term “cancer overexpressing EGFRvIII” refers to acancer having EGFRvIII on the cancer cell surface at a significantlyhigher level compared to non-cancerous cells of the same tissue type.

A pharmaceutically acceptable carrier to be contained in the compositionof the present disclosure is conventionally used in the formulation andincludes, but are not limited to, lactose, dextrose, sucrose, sorbitol,mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,water, syrup, methylcellulose, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and thelike. The composition of the present disclosure may further include,e.g., a lubricant, a wetting agent, a sweetener, a flavoring agent, anemulsifying agent, a suspending agent, and a preservative in addition tothe components.

The pharmaceutical composition of the present disclosure may beadministered orally or parenterally. The parenteral administration iscarried out by intravenous injection, subcutaneous injection,intramuscular injection, intraperitoneal injection, endothelialadministration, topical administration, intranasal administration,intrapulmonary administration, rectal administration, and the like.

Because a protein or peptide is digested when administered orally, acomposition for oral administration should be formulated to coat orprotect an active drug agent against degradation in stomach. Also, thepharmaceutical composition may be administered by any device which cantransport active substances to target cells.

The appropriate dosage of the composition according to the presentdisclosure may vary depending on factors such as the formulation method,the administration method, patient's age, body weight, sex, pathologicalcondition, food, administration time, route of administration, excretionrate and reaction sensitivity. Thus, a commonly skilled physician caneasily determine and prescribe a dosage that is effective for thedesired treatment or prophylaxis. For example, the daily dosage of thepharmaceutical composition of the present disclosure is 0.0001 mg/kg to100 mg/kg. The term “pharmaceutically effective amount” as used hereinrefers to an amount sufficient to prevent or treat cancer.

The pharmaceutical composition of the present disclosure may beformulated using a pharmaceutically acceptable carrier and/or anexcipient according to a method which can be easily carried out by thosehaving ordinary skill in the art to which the present disclosurepertains so as to be provided in a unit dosage form or enclosed into amulti-dose container. Here, the formulations may be in the form ofsolutions, suspensions or emulsions in oils or aqueous media, or in theform of extracts, grains, suppositories, powders, granules, tablets orcapsules, and may additionally include dispersing or stabilizing agents.

The composition of the present disclosure may be administered as anindividual therapeutic agent or in combination with another therapeuticagent, and may be administered sequentially or simultaneously with aconventional therapeutic agent.

EXAMPLES

Hereinafter, the present disclosure will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are for illustrativepurposes only and are not to be construed to limit the scope of thepresent disclosure.

Example 1: First Identification of Internalizing Antibodies UsingPatient-Derived Tumor Spheroids

To screen cells required for cell panning for identification ofanti-NRP1 antibody fragments, the NRP1 expression levels ofpatient-derived tumor spheroids obtained from the Institute forRefractory Cancer Research at Samsung Medical Center were analyzed byRNA-Seq followed by RPKM (Reads Per Killobase Million) method (FIG. 12),and cells with high expression level of NRP1 were selected by FACS(Fluorescence Activated Cell Sorting) method and used in cell panning.

scFv antibody fragments that bind to human NRP1 were identified by phagedisplay screening using the previously prepared synthetic scFv antibodyfragment phage library (Yang et al., Mol. Cells. 27:225-235, 2009). Eachof four sub-library samples was cultured in 400 ml culture medium(SB/ampicillin/2% glucose) for two hours to recover the phagemid vectorintroduced into Escherichia coli host cell ER2537 in a phage form. Whenthe absorbance at O.D. 600 reached about 0.5 to 0.7, the supernatant wasremoved by centrifugation at 5000 g for 20 minutes, and then the cellswere suspended in 400 ml of a secondary culture medium (SB/ampicillin).Then, 10¹² pfu (plaque forming unit) of a helper phage (VCSM13) wasadded to the cells and incubated for one hour. Next, 70 μg/ml ofkanamycin antibiotic (an antibiotic gene introduced in helper phage) wasadded and incubated overnight at 30° C. to allow the phage library to besecreted out of the host cells. Then, the culture obtained bycentrifugation was precipitated only in the form of phage usingpolyethylene glycol (PEG) solution, thereby obtaining a phage library.

The phage library obtained as described above and patient-derived tumorspheroids (4×10⁶) with high NRP1 expression were mixed, added to a totalof 5 ml of NBA (neurobasal medium), fixed in a rotator at 4° C., andthen rotated 360 degrees for 1 to 2 hours. Then, the cells werecentrifuged at 300 g for 5 minutes to remove the phage particles thatdid not bind to the patient-derived tumor spheroids, and then the cellswere washed again by adding 5 ml of NBA. This procedure was repeatedfour times. In the final step, patient-derived tumor spheroids andphages were placed in a T flask using 5 ml of NBA placed in an incubatorat 37° C., and were incubated for 30 minutes at 37° C. to induce thephage particles attached to the cell surface to enter the cells byinternalization.

Then, the cell solution was placed in a 15-ml conical tube, and thecells were separated by centrifugation at 300 g for 5 minutes, and thenwashed with 5 ml of cold PBS (phosphate buffered saline). The washingprocess was repeated 6 times. The number of these processes wasincreased as the number of the cell panning increased. Then, 5 ml of 0.1M glycine (pH 2.2) was added, and the mixture was kept at roomtemperature for 5 minutes to separate the cell surface-attached phageparticles from the cell surface. Then, the cells were separated bycentrifugation at 300 g for 5 minutes, and 0.5 ml of 100 mM TEA wasadded thereto. The cells were transferred to an e-tube and left at roomtemperature for 15 minutes. Next, the cell debris was separated bycentrifugation at 12,000 rpm for 5 minutes, and the supernatantcontaining the phage particles in the cells was collected andneutralized by mixing with 1 ml of 2M Tris (pH 8). Thereafter, theneutralized supernatant was placed in 8.5 ml of a culture medium (SB)containing pre-grown ER2537, and incubated at 37° C. at 120 rpm toinfect Escherichia coli host cell ER2537 with the phage particles.Thereafter, the culture medium was centrifuged at 3,000 rpm for 15minutes, and the precipitated ER2537 was mixed with 500 μl of a culturemedium (SB), followed by spreading on a 15 cm culture medium. Afterculturing, 5 ml of SB culture medium (50% glycerol) was added thereto,and the colonies were collected and stored (−80° C.) To proceed withrepeated cell panning, 1 ml of the stored phage solution from theprevious round of panning was taken and subjected to phage particleamplification. After incubation in host cell ER2537, the helper phagewas added, and the recovered phage particles were separated by PEGprecipitation. These particles were used for the next round of panningin the same manner. The third round of panning was performed, and thecell panning procedure is shown in FIG. 2. It was confirmed that theratio of the phage particles after the panning to those before thepanning was increased as the number of panning rounds increased. Thismeans that the internalized phage particles were amplified through cellpanning. The results are shown in Table 1 below.

TABLE 1 Cell panning using patient-derived tumor spheroids Input WashOutput Out/Input 1 round 1.1*10¹³ 2.2*10⁴  2.7*10³   2.5/10¹⁰ 2 round2.5*10¹³ 5.0*10³ 1.32*10⁵ 5.28/10⁹ 3 round 1.5*10¹³ 3.1*10⁴ 1.49*10⁶9.93/10⁷

Example 2: ELISA and Sequencing for Screening of Anti-NRP1 AntibodyFragment Candidates

The phage particles recovered from the 3rd round cell panning wereconfirmed as colonies in the medium through host cell (ER2537)infection. These colonies were taken, inoculated into 96-well platescontaining 200 μl of SB/ampicillin medium, and then incubated for 2 to 3hours at 37° C.

Then, each well was treated with IPTG (isopropylbeta-D-1-thiogalactopyranoside) at a final concentration of 1 mM forinduction of scFv-pIII protein expression and incubated overnight at 30°C. The incubated plate was centrifuged at 3,000 rpm for 15 minutes, andthe supernatant was removed. Thereafter, in order to recover the phageparticles from the periplasm of the incubated cells, 40 μl of TESsolution (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0) was added toeach well and left at 4° C. for 30 minutes so as to lyse the cells.Then, the cells were treated with 60 μl of 0.2×TES solution andincubated at 4° C. for 30 minutes to disrupt the cells by osmoticpressure. Then, the plate was centrifuged at 3,000 rpm for 15 minutes,and the supernatant scFv-pIII protein was obtained.

25 μl of the obtained supernatant was added to each of a 96-well platecoated with previously prepared human NRP1 protein, and then incubatedat room temperature for 1 hour, and each well was washed six times withTBST and distilled water. Then, each well was incubated withHRP-conjugated anti-HA antibody capable of binding to the HA tag ofscFv-pIII for 1 hour at room temperature, and then washed six times withTBST (0.1% Tween 20) and distilled water. TMB solution was used toinduce the color reaction. The color reaction was stopped with H₂SO₄solution, and the absorbance at 450 nm OD was measured.

The total number of clones analyzed was 384, of which 41 clones (bindingaffinity >2-fold) showed high binding affinity for human NRP1. As acontrol, BSA solution was used. Among the 41 clones, 10 clones with highbinding affinity were selected by ELISA. Then, the phagemid wasrecovered from 10 clones and subjected to DNA sequencing, and a total ofsix clones having different sequences were selected. Clones havingdifferent sequences were selected except for 3H10 having the samesequence as that of 1C08, and finally 3H10, 1A03 and 4F12 clones wereselected as anti-NRP1 antibody fragment candidates. The amino acidsequences of the 3H10, 1A03 and 4F12 clones are shown in Tables 2 and 3below.

TABLE 2  Heavy-chain FR/CDR sequences of anti-NRP1 antibody fragmentsFR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 1A03 EVQLLESGG GFTFS MSWVRQA ISFGYYADSVQGRFTISRD ARRKK WGQGT GLVQFGGSL SYY FGKGLEW SSNK NSKNTLYLQMNELRASFDY LVTVS RLSQAAS VSA EDTAVYYC S 3H10 EVQLLESGG QFTFS MSWVRQA ISPGYYADSVKGRFTISRD ARRKY WGQGT GLVQFQQSL SYY FGKGLEW SSNK NSKNTLYLQMNELRAMFDY LVTVS RLSQAAS VSA EDTAVYYC S 4F12 EVQLLESGG GFTFS MSWVRQA ISPGYYADSVKGRFTISRD AKRKT WGQGT GLVQFGGSL GYA FGKGLEW SGST NEKNTLYLQMNELRARFDY LVTVS RLSQAAS VSG EDTAVYYC S

TABLE 3 Light-chain FR/CDR sequences of anti-NRP1 antibody fragments FR1CDR1 FR2 CDR2 FR3 CDR3 FR4 1A03 QSVLTQFF SSNI VSWYQQ SCN NRPSGVPDRFSGGAWVA FGGGTKL SASGTPGQ GNND LFGTAP SKSGTSASLAIS SLSAY TVTL RVTISCSGKLLIY GLSSDEADYYC V F 3H10 GSVLTQPP SSNI VYWYQQ SQS NRSGMPDRFSG ASWDSFGGGTKL SASGTPGQ GNND LPGTAF SKSGTSASLAIS SLSGY TVTL RVTISCTG KLLIYGLRSEDEADYYC V E 4F12 QSVLTQFF SSNI VYWYQQ ANN KRFSGMPDRFSG AAWDSFGGGTKL SASGTFGR GNNE LPGTAP SKEGTSASLAIS SLNGY TVTL RVTISQSG KLLIYGLRSEDEADYYC V S

Example 3: Second Identification of Internalizing Antibodies UsingPatient-Derived Tumor Spheroids

The candidate antibodies recovered from the 3rd cell panning in Examplewere intratumorally injected into mice transplanted subcutaneously withthe patient-derived tumor spheroids used in Example 1.

After 20 hours, the mice were sacrificed, and the cancer tissue wasdissociated into single cells. Then, the antibody fragments internalizedin the cells were recovered and analyzed by ELISA in the same manner asdescribed in Example 2.

This in vivo cell panning procedure is shown in FIG. 3. It was confirmedthat the ratio of the phage particles after the panning to those beforethe panning was increased as the number of panning rounds increased.This means that the internalized phage particles were amplified throughcell panning. The results are shown in Table 4 below (unit: cfu/ml).

TABLE 4 Binding & no- Input internalization Output Output/Input 1 round2.4*10¹³  3*10⁶  8*10⁴ 3.3/10⁹ 2 round 3.5*10¹⁵ 1.8*10⁷ 1.2*10⁵ 3.4/10¹¹ 3 round 2.5*10¹⁵ 2.0*10⁸ 2.4*10⁵ 9.6/10⁹

Example 4: Production of Anti-NRP1 Antibody Fragments and Analysis ofBinding Affinity for NRP1

The basic structure of phagemid is shown in FIG. 4. In the case of thehost cell ER2537 used in the above examples, scFv cannot be expressedalone because the transcriptional suppression codon (amber codon (UAG))located upstream of phage pIII is suppressed. Therefore, using anexpression strain (TOP10F′) which is a non-suppressor strain, thephagemid was transfected into the expression strain. Thereafter, throughDNA sequencing, it was confirmed that the expression strain was anexpression strain in which each phagemid was introduced withoutmutation. The expression strain was taken as a colony, inoculated into 3ml of LB/ampicillin medium, and then cultured overnight at 37° C.Thereafter, 3 ml of the overnight culture was transferred to 400 ml ofmedium (SB/ampicillin) and cultured until the OD 600 reached 0.5 to 0.7.IPTG was added thereto to a final concentration of 1 mM, followed byculture overnight at 30° C. After the culture was centrifuged, 40 ml ofTES solution was used to lyse the expression host, and then 60 ml of0.2×TES was added and the phage particles in the periplasm wererecovered. The recovered supernatant was filtered through a 0.45 μmfilter. The scFv protein present in the filtered solution was added to 1ml of Ni-NTA bead (Qiagen) for His-tag purification and bound for 1 hourat room temperature. Thereafter, the resulting material was packed in agravity column (Bio-rad) and recovered via 200 mM imidazole solution.After the expression and purification of each clone, it was confirmedthrough SDS-PAGE and Coomassie blue staining that the size of scFv wasabout 28 kDa (FIG. 5).

Whether the purified scFv would have binding affinity for the targetNRP1 was analyzed by ELISA. In a 96-well plate coated with 200 ng ofNRP1 protein and a 96 well-plate coated with 200 ng of BSA as a controlgroup, binding was performed at a concentration of 5 μg/ml per eachclone at a room temperature for 1 hour by ELISA (3 times repetition).Thereafter, each well was washed three times with 0.1% TBST, treatedwith HRP-conjugated HA antibody for 1 hour, washed again, and thenincubated with TMB solution for 5 minutes. After the color developmentreaction was stopped with 2 M sulfuric acid solution, the OD value wasmeasured.

As a result, it was confirmed that 1A03, 3H10 and 4F12 scFv showedspecific binding affinity for NRP1, unlike 12B scFv that does not bindto NRP1 (FIG. 6).

Next, in order to measure the binding affinity of each antibody fragmentfor human NRP1 at varying antibody fragment concentrations, a 96-wellplate coated with 200 ng of NRP1 or BSA was treated with each scFv at aconcentration of 2,000 ng/ml, 1,000 ng/ml, 500 ng/ml, 250 ng/ml, 125ng/ml, 62.5 ng/ml, 31.25 ng/ml, or 15.62 ng/ml, and the change in the ODvalue was analyzed. Regarding the binding affinity for NRP1, the ODvalue of 12B scFv did not change depending on the concentration change.However, in the case of 1A03, 3H10 and 4F12 scFvs, it could be confirmedthrough the change in the OD value that the scFv bound to NRP1 increasedwith compared to BSA as the concentration increased (FIG. 7).

To accurately measure the degree of the binding affinity of the threescFv antibody fragments for NRP1 protein, the final KD value wasobtained through ka and kd values by using biacore T100, a surfaceplasmon resonance (SPR) system. The KD value is the value obtained bydividing the kd value by the ka value. The lower the KD value, thegreater the binding affinity for the corresponding substance. Theanalysis results indicated that 4F12 scFv showed the lowest KD (M) value(73.60×10⁻⁹), 1A03 scFv showed a KD (M) value of 89.40×10⁻⁹, and 3H10scFv showed a KD (M) value of 295.4×10⁻⁹ (FIG. 8).

Example 5: Analysis of Binding Affinity of Anti-NRP1 Antibody FragmentsUsing NRP1-Overexpressing Cell Line

After binding affinity for human NRP1 protein was analyzed by ELISA,FACS analysis was performed using patient-derived tumor spheroids withhigh expression of NRP1 in order to examine whether the antibodyfragment would bind to NRP1 present in the actual cell membrane. EachscFv was incubated with 5×10⁵ patient-derived tumor spheroids at 4° C.for approximately 1 hour, and then the cells were washed 3 times with 1ml of FACS solution. Then, the cells were treated with 1 μg of redfluorescence (PE; phycoerithrin)-labeled HA antibody and incubated at 4°C. for 30 minutes. Next, the cells were washed three times with 1 ml ofFACS solution and analyzed using a FACS Calibur™ system.

The results of the analysis indicated that the three antibody fragments,including 1A03, 3H10 and 4F12, all did bind specifically to theNRP1-overexpressing cell line compared to the cells treated withPE-conjugated HA antibody and 12B (FIG. 9).

Example 6: Analysis of the Penetration Ability of Anti-NRP1 AntibodyFragments into NRP1-Overexpressing Cancer Cells

The intracellular penetration abilities of the three anti-NRP1 antibodyfragments were analyzed by cell immunofluorescence staining. A PD-lysinesolution was added to a chamber slide and coated at room temperature for1 to 2 hours. The solution was removed and the slide was dried.Thereafter, the slide was treated with 200 μl of NBA solution containing5×10⁴ patient-derived tumor spheroids, and then incubated at 37° C. for4 to 5 hours to fix the cells to the slide. Next, the NBA solution wasremoved, and the cells were fixed in 4% paraformaldehyde at 4° C. for 10minutes. After washing three times with PBS, the cells were treated with0.1% Triton X-100 to increase cell penetration ability. In order tostain the NRP1 protein, the cells were treated with anti-human NRP1antibody (R&D) and anti-NRP1 antibody fragment at the same time andincubated at 37° C. for 15, 30 and 60 minutes. After washing three timeswith PBS, the cells were blocked with 1% BSA solution at roomtemperature for about 1 hour in order to block nonspecific binding. Assecondary antibody, green fluorescence (Alexa-Fluor 488)-labeled goatanti-mouse antibody (Invitrogen) was used to visualize the NRP1 protein,and anti-HA antibody (Santacruz biotechnology) was used to visualize theanti-NPR1 antibody fragment, followed by incubation at room temperaturefor 1 hour. Finally, DAPI staining was performed for nuclear staining.After final washing, the glass cover was fixed onto the slide which wasthen observed using a confocal laser scanning microscope.

As a result, it could be seen that, in all the three anti-NRP1 antibodyfragments, the anti-NRP1 antibody fragment attached to the cell surfaceand the anti-NRP1 antibody fragments inserted into the cells were mixedat 15 minutes and 30 minutes, but after about 60 minutes, the anti-NRP1antibody fragments were mostly inserted into the cells by penetration(FIGS. 10a to 10c ). In particular, the 4F12 antibody fragment exhibitedrelatively high cell penetration ability compared to 1A03 and 3H10 withthe passage of time (FIG. 10a ). These results show that the antibody ofthe present disclosure can be used for the purpose of delivering aprotein expression inhibitory substance or a therapeutic/diagnosticchemical drug into cancer cells.

Example 7: Transformation from Anti-NRP1 Antibody Fragment into NRP1 IgG

In order to transform the anti-NRP1 antibody fragment into an IgGformat, the heavy chain and light chain gene sequences of the NRP1antibody fragment were transfected using an Expi 293F expression system(life technologies). To recover the anti-NRP1 IgG antibody from theculture medium, purification was carried out using an AKTA proteinpurification system and an Amicon centrifugal filter. The amountsproduced were 120 mg/l for IRCR-101 (3H10 converted into IgG format), 66mg/l for A03, and 15 mg/l for 4F12. In order to confirm the purity ofthe purified anti-NRP1 IgG antibody, high performance liquidchromatography was used. Since IgG was 150 kD in size, the substancethat appeared at 16.388 min at the marker peak was IgG. Three anti-NRP1IgG antibodies (IRCR-101, 1A03, and 4F12) were detected at this peak,and showed purities of 99.5, 99.4, and 99.5%, respectively (FIG. 13).Limulus Amebocyte Lysate (LAL) QCL-1000™ kit was used to determineendotoxin levels of the three NRP1 antibodies produced. The analysisresults indicated that the three antibodies had an endotoxin level ofabout 0.5-3.1 EU/mg, which corresponds to the normal endotoxin level ofa therapeutic protein (FIG. 14).

The binding affinities of the three anti-NRP1 IgG antibodies for humanNRP1 were analyzed by ELISA and SPR analysis, and as a result, it wasconfirmed that the binding affinity was higher in the order of 1A03,IRCR-101 and 4F12. In particular, 4F12 had a KD value of 0.6 nM, whichis the binding affinity level of the current therapeutic antibody (FIG.15). The specific binding affinity for human NRP1 was analyzed bycomparison with other proteins having a structure similar to that ofhuman NRP1, and as a result, it was confirmed that all the threeanti-NRP1 IgG antibodies did bind only to human NRP1 (FIG. 16).

Example 8: Identification of Binding Epitope of Anti-NRP1 IgG Antibody

Since the binding domain of MNRP1685A is a VEGF domain, MNRP1685A wasused as a positive control. Each well of a 96-well plate was coated withhNRP1 protein, and then incubated with 500 nM of IRCR-101 or MNRP1685Aat 25° C. for 1 hour, washed with PBST, and then incubated withbiotin-conjugated VEGF or Sema3A at room temperature for 15 minutes.

The plate was washed with PBST, and then streptavidin-HRP antibody wasadded thereto and the TMB color development reaction was analyzed byELISA. As a result, it could be seen that MNRP1685A and IRCR-101 all didbind to the VEGF165 binding domain (the left panel of FIG. 11).

To identify the binding epitopes of the MNRP1685A and IRCR-101, eachwell of a 96-well plate was coated with 200 ng of hNRP1 protein, andthen incubated with 500 nM of IRCR-101 or MNRP1685A at 25° C. for 1hours, washed with PBST, and then biotin-conjugated IRCR-101 was treatedin MNRP1685A-treated wells and biotin-conjugated MNRP1685A was treatedin IRCR-101 treated wells at room temperature for 15 minutes.

The plate was washed with PBST, and then streptavidin-HRP antibody wasadded thereto and the TMB color development reaction was analyzed byELISA. As a result, it could be seen that the binding epitopes of thecontrol and IRCR-101 did differ from each other (the right panel of FIG.11).

Example 9: Analysis of Cancer-Specific Internalization and BindingAffinity Using Cancer Cells and Normal Cells

Internalization patterns of the three anti-NRP1 IgG antibodies intocancer cells and normal cells were compared using pHrodo® Red MicroscaleLabeling Kit (Thermo #p35363). According to the principle of the kit,when an antibody is conjugated with a chromogenic sample and theantibody is outside the cell, it does not develop color. On the otherhand, when the antibody enters the cell and the surrounding environmentis acidified, it develops color. According to this principle,intracellular internalization of the antibody can be confirmed.Conjugation to the three NRP1 IgG antibodies was performed, andinternalization patterns of the conjugated antibodies intopatient-derived cancer cells and normal HUVEC cells were compared. As aresult, internalized antibodies were started to be observed in thepatient-derived cancer cells from 20 minutes (FIG. 17).

Control IgG, IRCR-101, and 1A03 were injected in a glioblastomasubcutaneous model by an intravenous injection. After 20 hours, theywere sacrificed to separate into a single cell through celldissociation. Then immanence thereof to cancer cells were compared fromeach other using FACS. In the results of screening only antibodiesinternalizing into cancer cells through permeabilization, IRCR-101 and1A03 showed 5 times to 6 times higher mean fluorescence intensity (MFI)than the control IgG. It was also confirmed that the in vivo model hadcancer cells-specified immanence as in vitro model as described above(FIG. 18).

At a binding temperature of 4° C., the differences in the bindingaffinities of IRCR-101 and conventional NRP1 antibody (MNRP antibody,produced in house by synthesizing the sequence disclosed in the patent(WO2011143408)) for normal cells and cancer cells were compared. As aresult, it was confirmed that when the antibodies were used at the sameconcentration, the conventional NRP1 antibody showed a higher bindingaffinity for the normal cells than for the cancer cells, whereasIRCR-101 showed a specific binding affinity for the cancer cells (FIG.19).

Example 10: Conformation of Control of Cancer Cell Migration andDownstream Factor

Whether the three NRP1 IgG antibodies would inhibit cancer cellmigration examined using the glioblastoma cell line U87MG andpatient-derived tumor spheroids. After treatment with each antibody, thecells were incubated at 37° C. for 24 hours and then analyzed. As aresult, it was confirmed that IRCR-101 and 1A03 each showed more than50% cancer cell migration inhibition in the two types of cells and 4F12showed about 40% cancer cell migration inhibition in the patient-derivedtumor spheroids (FIG. 20).

The inhibition of migration of the breast cancer cell line MBAMB231 andthe lung cancer cell line A549 by the final anti-NRP1 IgG antibodyIRCR-101 was observed, and as a result, it was confirmed that theantibody inhibited cancer cell migration in a concentration-dependentmanner. It was confirmed that when the cells were treated with IRCR-101(10 μg/ml), the antibody showed 60% cancer cell migration inhibition inthe breast cancer model and 30% cancer cell migration inhibition in thelung cancer model (FIG. 21).

In order to examine a change in related signaling substances upontreatment with IRCR-101, changes in NRP1, AKT and ERK in glioblastomapatient-derived tumor spheroids at 15, and 120 min were analyzed byimmunoblotting. It was confirmed that NRP1 disappeared at 30 minutes dueto complete degradation and that AKT and ERK inhibited the relatedsignaling mechanisms since the phosphorylated AKT and ERK decreased(FIG. 22).

Example 11: Evaluation of Efficacy of IRCR-101 in In Vivo Models andObservation of Target

Two subcutaneous models were constructed using glioblastomapatient-derived tumor spheroids and injected intravenously with 5 mg/kgof IRCR-101, three times a week, and the size of the volume wasmeasured. The antibody showed 30-40% tumor size reduction, and the TUNELassay using immunofluorescence indicated that apoptosis was increased byIRCR-101 (FIG. 23).

Subcutaneous models were constructed using the glioblastoma cell lineU87MG and used to compare the efficacy of IRCR-101 with that of theconventional antibody MNRP1685A (MNRP1685A antibody, constructedin-house by synthesizing the sequence disclosed in the patent(WO2011143408 A1)). In the groups administered with 5 mg/kg of eachantibody twice a week, MNRP1685A showed 60% tumor growth inhibition, andIRCR-101 showed 80% tumor growth inhibition (FIG. 24).

Subcutaneous models were constructed using the lung cancer cell lineA549 and used to compare the efficacy of IRCR-101 with that of theconventional antibody MNRP1685A. In the groups administered with 25mg/kg of each antibody twice a week, MNRP1685A showed 19% tumor growthinhibition, and IRCR-101 showed 57% tumor growth inhibition (FIG. 25).

In glioblastoma orthotopic models, IRCR-101 was labeled with afluorescent substance and injected intravenously into the models, andthe time-dependent change in the fluorescence intensity was observed at15 min, 1 hr, 1 day and 2 day. As a result, it was confirmed that on day1, strong fluorescence appeared at the site corresponding to the tumorsite, and up to day 3, fluorescence appeared at the same site (FIG. 26).

Example 12: Evaluation of Distribution in Normal Tissues Using MonkeyTMA

In order to examine the side effects of IRCR-101 and the conventionalNRP1 antibody (MNRP1685A antibody, constructed in-house by synthesizingthe sequence disclosed in the patent (WO2011143408 A1)), TMA (TissuemicroArray) was performed using male and female monkeys. The results ofthe analysis indicated that IRCR-101 had little or no binding affinityfor most normal organ tissues, unlike the conventional NRP1 antibody.The fact that IRCR-101 has little or no binding affinity for normaltissues suggests that IRCR-101 will show less side effects in clinicaltrials (FIG. 27).

INDUSTRIAL APPLICABILITY

The screening method according to the present disclosure usespatient-derived tumor spheroids, and thus can screen an antibody thattargets a protein overexpressed specifically in a patient. The antibodyscreened according to the present disclosure may be used to produce apatient-specific antibody, and thus is useful for the development of apatient-specific therapeutic drug. In addition, the antibody orantigen-binding fragment thereof screened by the method of the presentdisclosure is expected to have a high possibility of success in futureclinical trials. Furthermore, the screening method according to thepresent disclosure can screen an internalizing antibody, and thus makesit possible to screen an antibody suitable for production of adrug-antibody conjugate (ADC).

Although the present disclosure has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present disclosure. Thus, thesubstantial scope of the present disclosure will be defined by theappended claims and equivalents thereof.

1. A method for screening an antibody or an antigen-binding fragmentthereof, which binds to an antigen, the method comprising the steps of:(i) treating patient-derived tumor spheroids, which express the antigen,with a library comprising antibodies or antigen-binding fragmentsthereof, and screening antibodies or antigen-binding fragments thereofwhich bind to the antigen; (ii) incubating the screened antibodies orantigen-binding fragments thereof with patient-derived tumor spheroidsthat do not express the antigen; and (iii) separating or removingantibodies or antigen-binding fragments thereof, which bind to thepatient-derived tumor spheroids of step (ii), from the antibodies orantigen-binding fragments thereof screened in step (i).
 2. A method forscreening an antibody or an antigen-binding fragment thereof, whichbinds to an antigen, the method comprising the steps of: (i) treatingpatient-derived tumor spheroids, which express the antigen, with alibrary comprising antibodies or antigen-binding fragments thereof, tofirstly screen antibodies or antigen-binding fragments thereof whichbind to the antigen; (ii) treating patient-derived tumor spheroids,which do not express the antigen, with the first screened antibodies orantigen-binding fragments thereof; (iii) administering antibodies orantigen-binding fragments thereof, obtained by separating or removingantibodies or antigen-binding fragments thereof that binds to thepatient-derived tumor spheroids of step (ii) from the antibodies orantigen-binding fragments thereof screened in step (i), to animal modelstransplanted with the patient-derived tumor spheroids which express theantigen, to secondly screen antibodies or antigen-binding fragmentswhich bind to the antigen; and (iv) separating or removing antibodies orantigen-binding fragments thereof, which bind to an antigen other thanthe antigen, from the second screened antibodies or antigen-bindingfragments thereof.
 3. The method of claim 1 or 2, wherein the antibodyor antigen-binding fragment thereof is internalized into cells.
 4. Themethod of claim 1 or 2, wherein the patient-derived tumor spheroidsexpressing the antigen are obtained by performing the following steps:(a) dissociating isolated cancer patient-derived cancer tissue, andcollecting a cell fraction from the dissociated tissue; and (b) treatingthe collected cell fraction with protease, followed by filtration,centrifugation and suspension, thereby obtaining single cells.
 5. Themethod of claim 2, wherein the animal models transplanted withpatient-derived tumor spheroids expressing the antigen areimmunodeficient mice.
 6. The method of claim 5, wherein theimmunodeficient mice are nude mice, NOD (non-obese diabetic) mice, SCID(Severe combined immunodeficiency) mice, NOD-SCID mice, or NOG (NOD/SCIDI12rg−/−) mice.
 7. The method of claim 1 or 2, further comprising a stepof converting the antibody or antigen-binding fragment thereof screenedby the method to an IgG format.
 8. An antibody or an antigen-bindingfragment thereof, which is screened by the method of claim 1 or
 2. 9. Acomposition for preventing or treating cancer, which comprises theantibody or antigen-binding fragment thereof of claim 8.